Ó Springer 2006
Plant Growth Regulation (2006) 48:97–109 DOI 10.1007/s10725-005-5481-7
Phospholipids as plant growth regulators A. Keith Cowan Nutra-Park Inc., Suite 140, 3225 Deming Way, Middleton, WI 53562, USA; (e-mail:
[email protected]) Received 17 August 2005; accepted in revised form 27 November 2005
Key words: Lysophospholipids, Phospholipids, Plant growth regulators
Abstract In this paper the potential to use phospholipids and lysophospholipids as plant growth regulators is discussed. Recent evidence shows that phospholipids and phospholipases play an important signalling role in the normal course of plant development and in the response of plants to abiotic and biotic stress. It is apparent that phospholipase A (PLA), C (PLC) and D (PLD), lysophospholipids, and phosphatidic acid (PA) are key components of plant lipid signalling pathways. By comparison, there is very little information available on the effect of exogenously applied phospholipids on plant growth and development. This paper serves to introduce phospholipids as a novel class of plant growth regulator for use in commercial plant production. The biochemistry and physiology of phospholipids is discussed in relation to studies in which phospholipids and lysophospholipids have been applied to plants and plant parts. Implicit in the observations is that phospholipids impact the hypersensitive response and systemic acquired resistance in plants to improve crop performance and product quality. Based on published data, a scheme outlining a possible mode of action of exogenously applied phospholipids is proposed.
Introduction Phospholipids are a major and vital component of all biological membranes and play a key role in processes such as signal transduction, cytoskeletal rearrangement, and in membrane trafficking. For example, phospholipids act as co-factors for membrane-localized enzymes involved in signalling cascades and facilitate protein–lipid and protein– protein interaction in which protein activation is achieved by either a lipid-induced conformational change or spatial rearrangement of the proteins within the lipid bilayer. Phospholipids and related catabolites (e.g. lysophospholipids) can change the physical properties of membranes to increase or decrease ion flux and membrane transport, vesicle formation, and endo- and exocytosis. Furthermore,
genetic studies using Arabidopsis thaliana confirm that changes in phospholipid homeostasis profoundly affect plant growth and development. Over-accumulation of phosphatidylinositol-4,5bisphosphate (PI4,5P2) and intositol-1,4,5-phosphate (IP3) is a characteristic of the sac9 mutants which show a constitutive stress response (Williams et al. 2005), a reduction in levels of phosphatidylglycerol (PG) in the pho1 mutant causes ultrastructural alterations in thylakoid organization (Ha¨rtel et al. 1998), and impairment of root hair tip growth in the srh1 (cow1) mutant is due to loss of an essential PI transfer protein (Bo¨hme et al. 2004). In recent years, the role of phospholipids in plant growth and development and the response of plants to biotic and abiotic stress has concentrated largely on the signalling mechanisms involved
98 (Chapman 1998; Laxalt and Munnik 2002; Meijer and Munnik 2003). Although the downstream targets and modes of action of lipid signals are not fully elucidated, it is apparent that phospholipase A (PLA), C (PLC) and D (PLD), lysophospholipids, and phosphatidic acid (PA) are key components of plant lipid signalling pathways (Munnik 2001; Wang 2001, 2004, 2005; Ryu 2004; Testerink and Munnik 2005). Natural and synthetic plant growth regulators are used in agriculture as management tools to improve crop performance and enhance product quality. Whereas the biochemical mode of action of many of these plant growth regulators is poorly understood and in some cases unknown, most are classified on the basis of their similarity in action to the naturally occurring plant hormones. Nevertheless, phospholipid metabolism and signalling are inextricably linked to the mechanism of action of plant hormones. A detailed analysis of the Arabidopsis thaliana genome has revealed the involvement of numerous polypeptides in phosphatidylinositol (PI) signalling (Lin et al. 2004). These include PI synthases, PI-phosphate kinases, PLCs, inositol polyphosphate phosphatases, inositol polyphosphate kinases, PI transfer proteins and putative inositol polyphosphate receptors (Mueller-Roeber and Pical 2002; Lin et al. 2004). Expression profile analysis of tissues treated with either auxin, cytokinin (CK), gibberellin, abscisic acid (ABA), brassinosteroid, salicylic acid or jasmonic acid (JA) or factors such as temperature, salinity, and drought confirmed that these PI pathway-related genes were differentially expressed but that various isoforms were in many cases expressed in a tissue and/or environmentspecific manner (Lin et al. 2004). PI-specific PLCs are also intimately involved in CK-mediated responses (Repp et al. 2004). Furthermore, auxininduced cell elongation is directly linked to PLA2 activity (Scherer 2002; Lee et al. 2003) and PLA2 is involved in JA biosynthesis (Ryu 2004) while ABA and ethylene-induced senescence have been associated with alterations in PLD activity (Fan et al. 1997). Phosphatidic acid, a major product of PLD activity, has recently been shown to occupy an important ABA signaling role during seed germination (Katagiri et al. 2005) while its immediate catabolite diacylglycerol pyrophosphate (DGPP) has been confirmed as a second messenger in ABA signal transduction pathways (Zalejski et al. 2005).
Changes in PLD activity are also linked to ABAinduced guard cell movement (Hallouin et al. 2002; Wang 2002), retardation of H2O2-induced cell death (Zhang et al. 2003) and CK signalling (Romanov et al. 2002). Thus, the link between phospholipid turnover and homeostasis and hormone action in plant growth and development is well established. This paper serves to introduce as a novel group of plant growth regulator, the phospholipids. Recent advances in our knowledge of the metabolism and function of endogenous phospholipids and phospholipid catabolites in plants has shown that this group of compounds is essential. Furthermore, an increasing body of evidence suggests that exogenously applied phospholipids and phospholipid derivatives exert profound effects on plant growth and development. In this article the possibility of using phospholipids as plant growth regulators is explored. First, a brief overview of current knowledge of the synthesis and breakdown of phospholipids is presented. Second, the response of plants and plant parts to exogenous phospholipids and lysophospholipids is highlighted. Finally, and based on data presented in the literature, a scheme outlining a possible mode of action of exogenously applied phospholipids is proposed.
Phospholipid metabolism and function Glycerophospholipids or phospholipids contain a polar phosphorus moiety (head-group) and a glycerol backbone which contains at least one Oacyl, or O-alkyl, or O-(1-alkenyl) group attached to the glycerol residue and this structural arrangement is illustrated in Figure 1. Broadly, phospholipids occupy both structural and signalling roles in plants and this bi-functional property is due in part to continued synthesis and turnover of the endogenous glycerophospholipid pools.
Phosphatidylphospholipids Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the major phospholipids in plant membranes and their pathway of biosynthesis is delineated in Figure 2. A minor pathway
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Figure 1. Structure of a phospholipid (a) and a lysophospholipid (b) and the associated head-groups. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.
Figure 2. Scheme illustrating the major steps in the biosynthesis of phospholipids in plants. CDP, cytidine diphosphate; CTP, cytidine triphosphate; DAG, diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP2, phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine.
to PC amine amine Other
involves methylation of phosphoethanolby the cytosolic enzyme phosphoethanolN-methyltransferase (McNeil et al. 2001). minor pathways include, conversion of
phosphatidylserine (PS) to PE (Rontein et al. 2003a), enzymatic reaction of ethanolamine with PS, or re-acylation of lysoPE (LPE). PS in plants can also arise by a pathway analogous to that
100 found in yeast (Delhaize et al. 1999) wherein PS is synthesized by the action of PS synthase. Some plasma membrane PS is synthesized in situ (Vincent et al. 1999) however the bulk is produced in the endoplasmic reticulum either by PS synthase or the serine Base Exchange enzyme (Marshall and Kates 1974; Moore 1975), and is then transported to mitochondria where it is decarboxylated to PE (Vincent et al. 2001). It is unknown why such a complex shuttle system is required in the synthesis of PE and PS but it may be necessary to coordinate membrane sterol requirements (Moreau et al. 1998). Alternatively, and as suggested by Voelker (2005) the formation of PE from PS transported into mitochondria and golgi might constitute a chemical reporter system mediating this transport process. Phosphatidylserine appears to be located entirely on the inner monolayer surface of the plasma membrane and externalization of PS is apparently an indication of apoptosis (O’Brien et al. 1998; Delhaize et al. 1999). In addition to its structural function, PS is an essential cofactor for the activation of protein kinase C (Karibe et al. 1995). Phosphatidylglycerol (PG) and PI, like PS, are minor components of membranes. In plants, PG and PI are formed from PA by a sequence of enzymatic reactions that proceed via cytidine diphosphate diacylglycerol (CDP-diacylglycerol), which reacts either with glycerol-3-phosphate or myo-D-inositol to give PG and PI respectively (Figure 2). Other minor routes to PG include PLD-catalyzed hydrolysis of diphosphatidylglycerol or glycerolysis of other phospholipids (also catalyzed by PLD). In chloroplasts, PG is the major phospholipid in thylakoid and envelope membranes and a defined level is indispensable for photoautotrophic growth (Frentzen 2004). Phosphatidylinositol is phosphorylated by a number of different kinases at positions 3, 4 and 5 of the inositol ring and the most important in both quantitative and biological terms are PI-4-phosphate and PI-4,5-bisphosphate. However, PI-3-P has recently been shown to play a role in ABA-induced reactive oxygen species (ROS) production and stomatal closure (Park et al. 2003). PI-phosphates are usually present at low levels, typically 1–3% of the concentration of PI and are maintained at a steady state level in the inner leaflet of the plasma membrane by continuous phosphorylation and dephosphorylation by
specific kinases and phosphatases, respectively. Hydrolysis of PI phosphates gives diacylglycerol (DAG), which also acts as a second messenger to regulate the activity of protein kinase C (PKC) in animal cells. Also released by this reaction is water-soluble inositol-1,4,5-phosphate (IP3), which is important in calcium and hormone signalling. The Arabidopsis genome indicates the absence of an IP3 receptor and PKC in plants (Meijer and Munnik 2003). Instead, plants seem to rapidly convert DAG to PA and PA-elicited signals are attenuated by phosphorylation of PA to diacylglyceropyrophosphate (DGPP), a molecule that is not present in animal cells (van Leeuwen et al. 2004). Furthermore, plants appear to use myo-inositol hexakisphosphate (IP6) rather than IP3 to mobilize intracellular Ca2+ stores (Lemtiri-Chlieh et al. 2003) in the control of key cellular functions such as differentiation, proliferation, metabolism and apoptosis.
Phospholipase and lyso phosphatidylphospholipids Catabolism of membrane phospholipids is essential in lipid-mediated signalling cascades and to maintain phospholipid homeostasis within the symplast. A steady-state phospholipid concentration is achieved by synthesis, catabolism, transport (vesicle trafficking), and sequestration (as structural components) and this metabolic interrelationship is illustrated in Figure 3. Lysophospholipids with one mole of fatty acid per mole of lipid are formed by hydrolysis of parent phosphatidylphospholipids by phospholipase A (PLA) and appear to play a significant role in mediating ATPase, phospholipase, and kinase enzymes (Palmgren et al. 1988; Palmgren and Sommarin 1989). Two types are associated with the PLA superfamily; phospholipase A1 (PLA1) and phospholipase A2 (PLA2) and these catalyze the hydrolysis of phospholipids at either the sn-1 or sn-2 position (Ryu 2004; Lee et al. 2005). PLA2 hydrolyzes the sn-2 acylester bond of membrane phospholipids to produce free fatty acids and 1acyl-2-lysophospholipids. Plant PLA2 enzymes are classified as either low-molecular-weight secretory PLA2 (sPLA2) or patatin-like PLA and the characteristics of each are summarized in Table 1. Patatin-like PLA is homologous to intracellular animal Ca2+-independent PLA2. No cytosolic
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Figure 3. Interconversion of membrane phospholipids and formation of lysophospholipids. AH, acyl hydrolase; BE, base-exchange enzyme; DAG, diacylglycerol; DGK, diacylglycerol kinase; LPP, lipid phosphate phosphatase; NAPES, N-acyl-PE synthase; N-acyl-E, N-acyl-ethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP2, phosphatidylinositol-4,5-bisphosphate; PIPK, phosphatidylinositol phosphokinase; PLA, phospholipases A; PLC, phospholipase C; PLD, phospholipases D; PS, phosphatidylserine; PSD, phosphatidylserine decarboxylase.
Table 1. Characteristics of PLA activities from plants. Enzyme
Ca2+requirement
MW
Substrate specificity
Function Jasmonic acid synthesis Senecsence
PLA1 PA-PLA1 Secretory-PLA2 (a, b , c, and d) PAT-PLA2=
lM-mM
13–18 kDa
sn-1 acyl group PC, MGDG PA sn-2 acyl group
mM
40–48 kDa
2-linolenoyl-PC
Cytosolic-PLA2
Not identified in plants
PLA2 has been identified in plants. Although the major cellular function could be regulation of the release of linolenic acid for JA synthesis, PLA2 has been reported to function in the following processes; germination and seedling growth, ROS generation and alkaloid production, auxin-mediated growth such as cell expansion, and responses to stress. It is believed that PLA2 is responsible for the formation of LPC, LPE, and LPI from PC, PE, and PI respectively. Lysophospholipids are present in membranes only in trace amounts but their concentration increases during acclimation to freezing (Welti et al. 2002), cell expansion (Scherer 2002) and in response to wounding (Lee et al. 1997). Thus, lysoPC was shown to activate the
Auxin signal transduction Peptide signal for secretion Acyl hydrolase activity=patatin; lyso-PLA activity
plant plasma membrane H+-ATPase, a key enzyme in cell expansion (Gome`s et al. 1996). Furthermore, the auxin response in plants was shown for the first time to involve PLA2 (for review see Scherer 2002). Further support for lysophospholipid involvement in auxin-mediated growth was obtained by showing that auxindependent hypocotyl elongation could be inhibited by inhibitors of animal PLA2 activity (Scherer and Arnold 1997). Moreover, transgenic plants overexpressing a secretory low-molecular-weight PLA2 displayed enhanced cell elongation whereas silencing of expression retarded cell elongation (Lee et al. 2003) seemingly confirming a regulatory role for PLA2 (and possibly lysophospholipids) in cell expansion.
102 N-acyl-phosphatidylethanolamine and N-acylethanolamine Both PE and LPE can be acylated in situ in the membrane in a process involving direct acylation of the amino group of PE/LPE with unesterified free fatty acids to form the corresponding N-acyl PE (NAPE) and this lipid species has been isolated from a variety of plant materials including wheat flour (Bomstein 1965), cereal seeds, other seeds, and is accumulated in plant tissues in response to stress (Dawson et al. 1969, De La Roche et al. 1973). NAPE has also been isolated from potato cells in amounts of 13 nmol/g fresh weight and its biosynthesis increases in response to anoxia about 10 h after onset of the stress stimulus (Rawyler and Braendle 2001). N-acyl PE is a minor lipid in plants that occurs in amounts
